Genetic characterization of 2 novel feline caliciviruses isolated from cats with idiopathic lower urinary tract disease

Feline caliciviruses (FCVs) are potential etiologic agents in feline idiopathic lower urinary tract disease (I-LUTD). By means of a modified virus isolation method, we examined urine obtained from 28 male and female cats with nonobstructive I-LUTD, 12 male cats with obstructive I-LUTD, and 18 clinically healthy male and female cats. All cats had been routinely vaccinated for FCV. Two FCVs were isolated; I (FCV-U1) from a female cat with nonobstructive I-LUTD, and another (FCV-U2) from a male cat with obstructive I-LUTD. To determine the genetic relationship of FCV-U1 and FCV-U2 to other FCVs. capsid protein gene RNA was reverse transcribed into cDNA, amplified, and sequenced. Multiple amino acid sequence alignments and phylogenetic trees were constructed for the entire capsid protein, hypervariable region E, and the more conserved (nonhypervariable) regions A, B, D, and F. When compared to 23 other FCV isolates with known biotypes, the overall amino acid sequence identity of the capsid protein of FCV-U1 and FCV-U2 ranged from 83 to 96%; identity of hypervariable regions C and E ranged from 58 to 85%. Phylogenetically, FCV-U1 clearly separated from other FCV strains in phenograms based on nonhypervariable regions. In contrast, FCV-U2 consistently segregated with the Urbana strain in all phenograms. Clustering of isolates by geographic origin was most apparent in phenograms based on nonhypervariable regions. No clustering of isolates by biotype was apparent in any phenograms. Our results indicate that FCV-UI and FCV-U2 are genetically distinct from other known vaccine and field strains of FCV.     

Quantitation of reticulated platelets in healthy dogs and in nonthrombocytopenic dogs with clinical disease

Background — Thrombocytopenia is a common disorder in dogs and development of an objective diagnostic assay to measure platelets newly released from bone marrow into the blood would provide a noninvasive way to predict megakaryocytopoiesis. Reticulated platelets are newly released platelets with increased concentrations of RNA that can be detected by flow cytometric analysis of blood stained with thiazole orange (TO).
Objectives — The goals of this study were to establish a reproducible method to quantitate reticulated platelets in dogs, to establish a reference interval for reticulated platelet percentages in healthy dogs, and to determine whether the percentage of reticulated platelets was nonspecifically increased in nonthrombocytopenic dogs with clinical disease.
Methods — Blood samples were obtained from healthy dogs and from nonthrombocytopenic dogs presented for a variety of disorders. An aliquot of whole blood was stained with TO and a phycoerythrin-labeled monoclonal antibody to platelet CD61, then analyzed by flow cytometry.
Results — The coefficients of variation were 7.8% to 15.6% (intra-assay precision) and 6.1% to 19.5% (interassay precision). Overnight storage for 18 to 26 hours, under variable conditions, resulted in an increase in the percentage of platelets staining with TO. The reference interval for reticulated platelets in the healthy control group was 0–4.3% (0–12,095/μL). No significant differences were found in the mean percentage of reticulated platelets or absolute concentration of reticulated platelets between control and affected dogs.
Conclusions — These studies demonstrate a reliable, noninvasive diagnostic assay for measurement of reticulated platelets in whole blood and provide a baseline for assessment of the clinical utility of the assay.     

Papillomavirus-associated basosquamous carcinoma in an Egyptian fruit bat (Rousettus aegyptiacus).

A 5-yr-old female Egyptian fruit bat (Rousettus aegyptiacus) had a small raised pigmented mass removed from the lateral canthus of the left eye. Six additional variably sized, raised, smooth to cauliflower-like skin masses were observed randomly distributed throughout the left wing membranes. Four masses were removed and diagnosed microscopically as basosquamous carcinomas and papillomas. Additional masses, removed 6 mo and 1 yr later, showed bony invasion and squamous differentiation. Immunohistochemistry detected positive intranuclear staining for bovine papillomavirus antibody in all samples. Polymerase chain reaction done on DNA extracts from formalin-fixed, paraffin-embedded tumor tissue amplified a 450 base-pair segment analogous to the L1 region of human papillomavirus types 96 and 5. Basic Local Alignment Search Tool analysis of sequenced amplicons suggests a novel chiropteran papillomavirus. To our knowledge, this is the first report of papillomavirus-associated carcinoma in a chiropteran species.     

Drought responses of six ornamental herbaceous perennials

Although low water use landscaping is becoming common in arid regions, little is known about drought tolerance and drought responses of many ornamental plants, especially herbaceous perennials. Drought responses were assessed for six herbaceous ornamental landscape perennials in a 38 l pot-in-pot system in northern Utah over a 2-year period. The first year was an establishment period. During the second year, drought responses were evaluated for established Echinacea purpurea (L.) Moench, Gaillardia aristata Pursh, Lavandula angustifolia P. Mill., Leucanthemum × superbum (J.W. Ingram) Berg. ex Kent, ‘Alaska’, Penstemon barbatus Roth var. praecox nanus rondo, and Penstemon × mexicali Mitch. ‘Red Rocks’. Plants were irrigated at frequencies of 1 (control), 2, or 4 weeks between June and September, simulating well-watered conditions, moderate drought, or severe drought. Osmotic potential (Ψ_s), gas exchange, visual quality, leaf area, and dry weight were assessed. In a confined root zone, P. barbatus showed the greatest tolerance to all levels of drought, avoiding desiccation by increasing root:shoot ratio and decreasing stomatal conductance as water became limiting. L. angustifolia and P. × mexicali showed tolerance to moderate drought conditions, but died after exposure to the first episode of severe drought. Neither G. aristata nor L. superbum were able to regulate shoot water loss effectively. Instead, both species displayed drought avoidance mechanisms, dying back when water was limiting and showing new growth after they were watered. Compared to control plants, G. aristata shoot dry weight was reduced by 50% and 84%, and L. superbum shoot dry weight was reduced by 47% and 99% for the 2- and 4-week irrigation intervals, respectively. Root dry weights were affected similarly for both species. E. purpurea exhibited poor visual quality at all irrigation intervals, in particular wilting severely in both drought treatments, but regaining turgor when watered again. P. barbatus is recommended for ornamental landscapes that receive little or no supplemental irrigation, while E. purpurea is not recommended for low water landscapes because of low visual quality under even mild drought.     

Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.     

南方菜豆花叶病毒(SBMV)两典型株系特异cDNA和RNA探针的制备及应用

南方菜豆花叶病毒（ＳＢＭＶ）主要有２株系：菜豆株系（ＳＢＭＶ－Ｂ）和豇豆株系（ＳＢＭＶ－Ｃ）,血清学方法不能予以区分。根据已报道的核酸序列设计了２对特异引物,进行ＲＴ－ＰＣＲ扩增并克隆到载体Ｂｌｕｅｓｃｒｉｐｔ中,序列测定予以证实后利用随机引物法合成放射性ｃＤＮＡ探针,再进行体外转译合成地高辛ＵＴＰ标记的ＲＮＡ探针。２种探针均可分别特异地检测Ｎｏｒｔｈｅｒｎｂｌｏｔ膜上的ＳＢＭＶ－Ｂ和ＳＢＭＶ－Ｃ。ＲＮＡ探针检测ＳＢＭＶ－Ｂ和ＳＢＭＶ－Ｃ灵敏度分别为：０．１μｇ和０．０１μｇ。